Methods for reducing inflammatory reactions using certain s-triazolo-(3,4-a)-isoquinolines

ABSTRACT

A number of mono- and poly- substituted s-triazolo-(3,4-a)isoquinolines are shown to have antiinflammatory/analgesic activity, and, in some instances, are additionally shown to have anti-secretory activity. The 3-trifluoromethyl derivative is preferred.

United States Patent [191 Cavallito et a1.

[451 Mar. 25, 1975 I 1 METHODS FOR REDUCING INFLAMMATORY REACTIONS USING CERTAIN S-TRIAZOLO-(3,4-A)-ISOQUINOLINES [75] Inventors: Chester John Cavallito, Greenwich,

Conn.; Allan Poe Gray, Essex Junction, Vt.

[73] Assignee: Mallinckrodt Chemical Works, St.

Louis, Mo.

[22] Filed: Apr. 23, 1973 [21] App]. No.: 353,696

Related US. Application Data [63] Continuation of Ser. No. 140,198, May 4, 1971,

abandoned.

[52] US. Cl. 424/258, 424/248 [51] Int. Cl A6lk 27/00 [58] Field of Search 424/258, 248

[56] References Cited UNITED STATES PATENTS 3,354,164 11/1967 Francis 260/288 FOREIGN PATENTS OR APPLICATIONS 1,573,135 7/1969 France 260/283 OTHER PUBLICATIONS J. Heterocyclic Chem. 3, 158-164 (1960).

Primary ExaminerAlbert T. Meyers Assistant Examiner-Leonard Schenkman Attorney, Agent, or FirmKoenig, Senniger, Powers and Leavitt [57] ABSTRACT 13 Claims, 2 Drawing Figures umas m5 SHEET 2 U? 2 NOE ow ow oo 0 91 8. 02 com 8m 2% (EHOSSBHCI SWVHQ) METHODS FOR REDUCING INFLAMMATORY REACTIONS USING CERTAIN S- TRIAZQLQ Q,4-a)-IS()QUINOLINES This is a continuation of application Ser. No. 140,198, filed May 4, 1971, now abandoned.

BACKGROUND OF THE INVENTION The present invention relates to the drug field, and more particularly to methods and pharmaceutical compositions for treating mammals utilizing as active agents certain synthetic organic heterocyclic compounds from the class known as striazolo-[3,4-a]- isoquinolines.

The preparation of various s-triazolo-l 3,4-alisoquinolines is disclosed by l S. Naqui et 211., Indian J. Chem, 3, 162-4( 1965); (2).G. S. Sidhu et al., Jour. Hererocyclic Cllem., 3, 158-l64 (1966); (3) J. E. Francis, U.S. Pat. No. 3,354,164 (1967); (4) H. K. Reimlinger et al., French Pat. No. 1,573,135 (1969) and (5) H.

K. Reimlinger et al., Chem. Ber. 103, 19601981 No pharmaceutical compositions or utility are disclosed in references (1), (2), (4) and (5) cited above. Francis (3) discloses that unsubstituted s-triazolo-[3,4- al-isoquinoline and its 3-lower alkyl derivatives are coronary vasodilators.

The invention is particularly concerned with the treatment of inflammatory disorders, among which are the conditions known as arthritis.

The term arthritis is applied to a group of related disorders of the joints which are characterized by pain, inflammation and stiffness. Certain types of arthritis are due to specific infectious agents, but no specific cause is known for the most common type, known as rheumatoid arthritis. While this is primarily an inflammatory disease of the joints, it is sometimes accompanied by pedal edema, that is, an accumulation of fluid in the tissues of the foot. Treatment of this disease involves the administration of agents which have been found effective in reducing the pain and the inflammation. Such agents have come to be generally known as anti-inflammatory agents.

Chemical compounds having structures of varying complexity, from the structurally simple aspirin to relatively complex steroid compounds, are known to be useful anti-inflammatory agents in the treatment of arthritic and related disorders in man and other mammals. Among such recognized anti-inflammatory agents, in addition to aspirin, are the following, to which reference may be conveniently made in the Merck Index, 8th Ed., Merck & Co., Rahway, N. J. (1968): dexamethasone (p. 334), hydrocortisone (p. 542) indomethacin (p. 566), mefenamic acid (p. 648) and phenylbutazone (p. 815), No s-t riazolo-[3,4-a]- isoquinolines are among the compounds which have heretofore been recognized as having antiinflammatory activity.

Detection of anti-inflammatory activity is a relatively difficult matter. Many unsuccessful attempts have been made to produce a disease in experimental animals analogous to rheumatoid arthritis. Several procedures have been developed, utilizing induced disorders in animals, to evaluate anti-inflammatory activity on a comparative basis. Among the established procedures for this purpose are the following:

1. lnhibition of Carrageenin-Induced Pedal Edema Winter et al., Proc. Soc. Exptl. Biol. Med. 111: 544

(1962). After administration of the drug a solution of carrageenin is injected into the left hind paw of the test animal, and the volume of edema is calculated from the increase in volume of the paw.

2. lnhibition of Cotton Pellet Granuloma Meier et al., Experentia 6: 469 (1950). A sterile cotton disc is implanted subcutaneously into a rat, with concurrent oral administration of the drug twice daily for four days. The inflammation due to the presence of the cotton causes formation of a granuloma. Five days after implantation the pellet and granuloma are removed. The increment of dry weight is taken as the measure of granuloma formation.

3. lnhibition of Thermal Edema Rocha e Silva et a1., Med. Exp. 3: 371 (1960). The drug is administered orally to a rat prior to immersion of the left hind paw in water at 55C. for 30 seconds. Edema caused by the heat increases the paw volume, which is measured at intervals.

4. Freunds Adjuvant Studies (See generally Newbould, Brit. J. Pharmacol. 21: 127 (1963). The drug is administered orally, in accordance with the procedure described hereinafter, to combat the arthriticlike syndrome produced by injection of Mycobacteriunz butyri cum (Freunds adjuvant).

Just as the inflammation of rheumatoid arthritis is accompanied by pain, it has been observed that antiinflammatory activity is frequently closely associated with analgesic (pain-relieving) activity.

Complementing the test procedures outlined above, a corresponding group of animal tests to assess analgesic activity has also been developed. These include the following:

1. Pressure-Pain Method Randall and Selitto, Arch. Int. Pharmacodyn. l l l: 409 (1957). Rats are injected in the left hind paw with a suspension of brewers yeast. This causes an inflammation which increases the sensitivity to pain caused by pressure on the foot. This sensitivity can be modified by analgesic agents.

2. Anti-writhing Vander Wende et al., Fed. Proc. 15: 494 (1956). lntraperitoneal injection of phenyl-pquinone in mice cause acute abdominal pain characterized by numerous writhing episodes (contraction and torsion of the abdomen). Administration of an analgesic prior to the administration of the phenyl-p-quinone modifies the number of writhing episodes occurring within a standard time interval, and this is used as an index of analgesic potency.

3. Rat Tail Flick DAmour and Smith, J. Pharmacol. Exptl. Therap. 72: 74 1941 Differences in the sensitivity of a rats tail to heat before and after drug administration is utilized as an index of analgesic potency.

SUMMARY OF THE INVENTION Among the several objects of the invention may be noted the provision of useful pharmaceutical compositions and methods for combatting an inflammatory reaction in a susceptible mammal; the provision of such compositions and methods which involve the use of certain s-triazolo-[3,4-a]isoquinoline compounds; the provision of compositions of the character indicated which exhibit anti-inflammatory/analgesic activity and which, in some instances, additionally exhibit antisecretory activity; and the provision of such compositions which are adapted for either oral or parenteral administration to mammals susceptible to inflammatory reactions. Other objects and features of the invention will be in part apparent and in part pointed out hereinafter.

The present invention is thus directed to a method of combatting an inflammatory reaction in a susceptible mammal which comprises administering to said mammal an effective amount of an s-triazolo-l 3,4-alisoquinoline compound from the group hereinafter specifically set forth. The invention is also directed to pharmaceutical compositions comprising such a compound and a pharmaceutical carrier.

BRIEF DESCRIPTION OF THE DRAWING FIG. 1 is a graphical representation showing the effect of 3-trifluoromethyl-s-triazolo-[3,4-a]- isoquinoline on adjuvant-induced polyarthritis; and

FIG. 2 is a graphical representation showing the analgesic activity of 3-trifluoromethyl-s-triazolo-[3,4-a]- isoquinoline and other agents on yeast inflamed rat paws.

DESCRIPTION OF THE PREFERRED EMBODIMENTS In accordance with the present invention, it has now been found that certain s-triazolo-[3,4-a]-isoquinoline compounds are useful anti-inflammatory agents. In this series of compounds, anti-inflammatory activity has been found to be quite selective. In other words, the presence or absence of such activity, and its degree when present, is quite sensitive to the position and type of substitution on the basic s-triazolo-[3,4-a]- isoquinoline structure.

The specific s-triazolo-[3,4-a]-isoquinoline compounds which have been found to possess antiinflammatory activity are:

3-trifluoromethyl-s-triazolo-[ 3,4-a ]-isoquinoline 6-chloro-s-triazolo-l3,4-a]-isoquinoline 3-(N-morpholinylmethyl)s-triazolo-l3,4-a]- isoquinoline 5-chloro-s-triazolo-[3,4-a]-isoquinoline 7-nitro-s-triazolo-[3,4-a]-isoquinoline 6-cyano-s-triazo1o-[3,4-a]-isoquinoline 7-chloro-s-triazolo-l3,4a]-isoquinoline 6-bromo-s-triazolo-[ 3,4-a1-isoquinoline 6-methoxy-s-triazolo-[3,4-a]-isoquinoline 7-nitro-3-trifluoromethyl-s-triazolo-[ 3 ,4-a]- isoquinoline 3-chloro-s-triaz0lo-l 3,4-al-isoquinoline 3 ,6-dichloro-s-triazolo-l 3,4-a l-isoquinoline 3-styryl-s-triazolo-l 3 ,4-a ]-isoquinoline 3-methylthio-s-triazolo-l 3,4-a]-isoquinoline 3-methyl-6-chloro-s-triazolo-l3,4-a]-isoquinoline 3-ethylthio-s-triazolo-l 3 ,4-al-isoquinoline 8-methoxy-s-triazolo-[3,4-a]-isoquinoline 6-chloro-3-isopropyl-s-triazolo-[3,4-al-isoquinoline 3-methyl-b bromo-s-triazolo-[3,4-a]-isoquinoline 7-bromo-s-triazolo-[ 3 ,4-a]-isoquinoline 3-bromo-s-triazolo-[3,4-a]-isoquinoline 3-phenyl-s-triazolo-l3,4-a]-isoquinoline 3-methyl-s-triazolo-[3,4-a]-isoquinoline s-triazolo-l3,4-a]-isoquinoline 3-oxo-s-triazolo-[3,4-a]-isoquinoline (s-triazolo-[3,4-a]-isoquinoline -3(2H)-one) 3-thioxos-triazolo-[3,4-a]-isoquinoline (s-triazolo-[3,4-a]-isoquinoline-3(2H)-thione) and the pharmaceutically acceptable, nontoxic acid addition salts thereof. Such addition salts may be derived from hydrochloric acid, hydrobromic acid. phosphoric acid, methanesulfonic acid and the like.

Homologous compounds, such as 3-ethyl-s-triazolo- [3,4-a1-isoquinoline, 6-chloro-3-ethyl-s-triazolo-l3,4- a]-isoquinoline and 6-bromo-3-isobutyl-s-triazolo-l 3,4- a]-isoquinoline and the aforementioned salts thereof. also possess anti-inflammatory activity.

Consistent with previous knowledge, in many of the above-noted compounds, analgesic activity is desirably associated with the anti-inflammatory activity. Further, certain of the compounds (e.g., B-trifIuoromethyI-striazolo-l 3,4-aJ-isoquinoline, 6-chloro-s-triazolo-l 3 ,4- aJ-isoquinoline, 7-nitro-s-triazolo-[3,4-al-isoquinoline, 3-methylthio-s-triazolo-[3,4-a]-isoquinoline and 3 ethylthio-s-triazolo-l3,4-a]isoquinoline) advantageously exhibit anti-secretory activity rather than the ulcerogenic properties exhibited by certain prior art anti-flammatory agents, such as the cortical steroids.

In the preferred embodiment of the invention, 3- trifluoromethyl-s-triazolo-l3,4-a1-isoquinoline is em ployed as an anti-inflammatory agent. As will appear from the detailed test results set forth hereinafter, this compound is an effective anti-inflammatory/analgesic agent active in a variety of pharmacologic procedures. Summarizing its properties, this compound is effective against carrageenin-induced pedal edema in both intact and adrenalectomized rats and in guinea pigs. In other animals, the compound shows activity in the cottonpellet granuloma assay in both intact and adrenalectomized rats, appears capable of inhibiting the onset of thermally-induced edema, and is active locally while being devoid of counterirritant effects. The compound significantly inhibits adjuvant-induced polyarthritis. It does not produce thymolysis, adrenal hypertrophy or gastric irritation. Further, it reduces yeast-induced fever in rats, phenylquinone-induced writhing in mice. and elevates yeast-induced pain threshold in rats. The compound causes little or no gross pharmacological symptoms in higher species (dog, monkey) and appears devoid of serious autonomic and cardiovascular effects. In lethality studies, the compound shows a higher therapeutic safety margin than any of the standard reference agents tested. Finally, the compound appears non-anticholinergic and significantly reduces gastric acid secretion by several routes of administration utilizing the pyloric-ligated rat procedure.

In accordance with the invention, pharmaceutical compositions therapeutically useful for combatting inflammatory reactions in susceptible mammals are pro vided and comprise an aforementioned s-triazolo-[ 3,4- a]-isoquinoline compoundand a pharmaceutical carrier which may be either liquid or solid material. These compositions may beadministered orally or parenterally in the usual pharmaceutical forms including capsules, tablets, solutions, suspensions and the like. For example, the s-triazolo-[3,4-al-isoquinoline compound may be formulated with carriers such as magnesium stearate and lactose and filled into gelatin capsules. Examples of other solid pharmaceutical carriers, such as fillers, binders and lubricants, include dibasic calcium phosphate, calcium sulfate dihydrate, microcrystalline cellulose, calcium carbonate and talc. The pharmaceutical compositions of the invention may also be in the form of sterile parenteral solutions with the s-triazolo- [3,4-a]-isoquinoline compound dissolved in a sterile parenteral solvent such as polyethylene glycol, propy lene glycol, water or mixtures of solvents or the compositions may be in the form of suspensions.

Where the s-triazolo-l3,4-a]-isoquinoline compound is water-insoluble (e.g., 3-trifluoromethyl-s-triazolo [3,4-a]-isoquinoline), it is preferred that the compound be formulated into the pharmaceutical compositions of the invention in a micronized form, as by milling the compound by conventional methods. More particularly, it is preferred that the compound be micronized to a particle size of approximately l-lO microns.

The following examples illustrate the invention:

EXAMPLE 1 3-Trifluoromethyl-s-triazolo-[3,4-al-isoquinoline (hereinafter designated NC-ll40) (French patent No.

1,573,! 35) was milled to pass a No. 325 US. Standard Sieve. The milled product (100 g.) was thoroughly blended with magnesium stearate (2 g.) and lactose (298 g.). Size No. l gelatin capsules 1000) were filled with the mixture. Each capsule contained 100 mg. of NC-] 140.

Similarly, capsules containing 10, 25, 37.5, 50 and 200 mg. of NC-l 140, respectively, were prepared.

in the formula described above, the lactose may be wholly or partially replaced by any of the following: dibasic calcium phosphate, calcium sulfate dihydrate, microcrystalline cellulose, calcium carbonate. or talc.

For studies in mice, rats and guinea pigs, aqueous suspensions containing the desired dose of finely milled (-325 mesh) NC-l 140 and 0.25 percent methylcellulose were prepared. A similar preparation containing NO] 140 (20 mg./ml.) and methylcellulose (0.5 percent W/V) was prepared and used for studies in monkeys.

NCl I40 is a white, crystalline material which is insoluble in water. Unlike a majority of non-steroid antiinflammatory agents, NC-l 140 has no acidic function. For most of the animal studies described below, the compound was utilized in a micronized form (particle size ll u) and administered as a finely divided suspension in a 0.25 percent methylcellulose solution to mice, rats, and guinea pigs. Rats (male Wistar, 100 to 175 grams) and mongrel dogs of both sexes were fasted for approximately 18 hours prior to use. The compound was administered orally in capsules to dogs. Monkeys were given the agent by intubation utilizing a 0.5 percent methylcellulose vehicle. For pharmacodynamic and autonomic studies in cats and dogs, NO I 140 was dissolved in polyethylene glycol 200 or 400.

EXAMPLE 2 Anti-Inflammatory Activity of NC-1l40 A. Inhibition of Carrageenin-lnduced" Pedal Edema (Rats) The procedure used for measuring inhibition of carrageenin-induced edema was a modification of the method of Winter et al., Proc. Soc. Exptl. Biol. Med. 111: 544 (1962). The device used for measurement of the paw volume was an adaptation of the Water displacement procedure described by Adamkiewicz et al., Can. J. Biochem. Physiol. 33: 332 (1955). NC-l 140 was studied and compared with selected antiinflammatory drugs in preventing the edema caused by the intraplantar injection of 0.05 ml. of a sterile 1.0 percent solution of carrageenin. All drugs were administered (orally or intraperitoneally) 1 hour prior to the injection of the phlogistic substance into the left hind paw of rats. At peak swelling time (3 hours), the volume of edema was calculated by differential paw volumes. The ED value was obtained for each drug and is defined as that does which reduced edema formation by percent or more compared with the mean control response (parallel run) in 50 percent of the animals. The following results were obtained:

Table 1 Effect of Selected Anti-inflammatory Agents on Carragecnin-lnduced Paw Edema (Rats) ED. mg.kg.('-)5'/ Confidence Limits) Drug Oral LP.

Aspirin 224 102-490) 16 10-26) Phcnylbutazone (l t-62) 9.8 5.4-l7.6)

Hydrocortisone 26 (13-55) 9.3 5.6-l5.5)

Acetate NC-ll 2l (ll-40) ll.2 (bl-20.7)

lndomethacin 2.4 (1.1-4.9) 0.5 (0.4-0.7)

Dexamethasone 0.18 (016-020) 0.1] (0.074). [8)

Thus, NC-l I40 is approximately equipotent, on a Table 2 Effect of Various Agents on Reducing Established Edema Oral Dose 76 Reduction in Established Edema Drug (mg/kg.) 1 hour 3 hours Aspirin 25 0 0 O 26 100 0 42 200 0 64* NC-l l40 25 33 56 50 39** 100 37" 69" 200 o6 Phenylbutazone 1O 17* Q 20 l6 I7 40 l5 I9 32** Hydrocortisone 5 0 0 Acetate 10 1 I0 20 3 [7* 40 i2 28 Dexamethasone 0.5 0 3 0 0 2 0 l2 7 v 4 Y 0 16* Mefenamic Acid 10 3 0 20 0 0 40 i5" 6 80 0 0 lndomethacin 0.3 0 0 1 0 0 3 0 0 l0 0 0 *P s .05 al 5-01..

The data indicate that NC-l 140, aspirin, phenylbutazone, hydrocortisone acetate, and, to a lesser extent. dexamethasone were capable of reducing previously established rat hind paw edema induced by carrageenin. Both mefenamic acid and indomethacin showed little or no activity in this procedure. NC-l [40, at all dose levels tested, appears far more effective at both the one and three hour measurements than any of the. other agents studied.

C. Inhibition of Carrageenin-Produced Edema (Guinea Pigs) To demonstrate anti-inflammatory activity in another species, inhibition of carrageenin-produced pedal edema was also studied in guinea pigs at selected dose levels with several representative compounds. The results given in the following table indicate that NC- 1140, phenylbutazone, and, to a lesser extent, indomethacin, at the doses studied. were active in this spe- D. Cotton Pellet Granuloma Inhibition of granuloma formation was determined by a modification of the method of Meier et al., Experentia 6: 469 (1950). Essentially it consisted of the subcutaneous implantation of a sertile cotton disc into either intact or adrenalectomized rats with concomitant oral administration of drug twice daily for four days. Removal of the pellets along with the granuloma formation after five days was performed and the increment in dry weight considered as the measure of granuloma formation. Based on several studies, a 40 percent reduction in granuloma formation was considered significant. Table 4 shows the'ED values obtained with NC-l 140 and several standard reference agents.

The results indicated that anti-inflammatory activity can be displayed in both normal and adrenalectomized rats. This implied that the anti-inflammatory activity of N01 140 was not caused by the release of endogenous adrenocortical steroids.

Table 4 Effect of Various Anti-Inflammatory Agents in the Cotton Pellet Granuloma Procedure ED vs. Cotton Pellet E. Thermal Edema Inhibition of thermally-induced edema was studied and adapted from the method of Rocha e Silva et al., Med. Exp. 3: 371 (1960). The drugs were administered orally to rats 30 minutes prior to immersionof the left hind paw in water at 5 5fC. for 30 seconds. The volume of resultant edema was measured 1 and 4 hours after immersion.

Table 5 Effect of Various Anti-Inflammatory Agents on Thermally-induced Edema With the experimental method used it appears that both aspirin and NC-l were capable of delaying the onset of thermally-induced edema (1 hour) but did not prevent or inhibit the eventual massive swelling of the paw (4 hours). lndomethacin did not appear effective in inhibiting edema at either time after paw immersion. F. Freund Adjuvant Studies (polyarthritis) NC-l 140 was utilized to combat the arthriticlike syndrome produced by injection of Mycobacterium butyricum (Freunds adjuvant).

1. Procedure a. Four groups of 10 rats each were used.

Group 1 These rats received no adjuvant injection and were orally dosed daily with the methylcellulose suspending agent. These control animals served to show the increase in paw size through normal body growth over the 31- day experimental period.

Group 2 This group was injected in the tail with the adjuvant and orally dosed daily with methylcellulose. The purpose of this group was to show the progress of the arthritic state without an attempt to alter the syndrome.

Group 3 These animals received the adjuvant injection on day 1 and were dosed orally with methylcellulose for the first 14 days of the experiment. On day 15, the animals were dosed orally with NC-1l40 (25 mg/kg.) and were continued on this regime for the remaining 15 days. This treatment was an attempt to reverse or arrest the mechanisms responsible for pro ducing the arthritic syndrome which were postulated to become evident at this point in the experiment.

Group 4 This group was injected with the adjuvant on day l and were dosed orally with 25 mg/kg/day of NC-l140 for 30 days. This group represented an attempt to prevent the development of the arthritic syndrome.

Dorso-ventral measurement of the hind paws and lateral measurements of the tibiotarsal joints were made with precision calipers on days 1, 15 and 31. Increased thickness of these areas over the nonadjuvant, no drug controls (Group 1) was considered a manifestation of the polyarthritic syndrome. In addition, visual inspection was made on the final day for the appearance of secondary lesions on the tail and skin surface of the paws. Other parameters measured on the final day were body weight gain, adrenal gland weights, thymus gland weights and gross inspection of the stomach linings for possible ulcerogenesis.

2. Results FIG. 1 illustrates the results obtained with the two measurements of paw and joint thickness. On day l the thickness of both the dorso-ventral and tibiotarsal areas showed little inter-group deviation indicating similar base line values for both of; these parameters. By day l there were no significant changes from normal growth indicating no evidence for the onset of the arthritic state. By day 3l. however, the measurements of paw and joint thickness in Group 2 (adjuvant and no drug) were significantly (P 2 .05) higher than Group 1 (no adjuvant and no drug)indicating that swelling did occur. Measurements in Groups 3 and 4 (receiving NC-l 140) were significantly. lower than Group 2, indicating that the drug was showing an effect in both arresting and inhibiting the arthritic'like condition. No internal toxicity was observed.

EXAMPLE 3 Analgesic Activity of NC-l 140 A. Pressure-Pain Method lntraplantar injection of brewers yeast in the hind paw of rats causes an inflammation which increases the sensitivity to pain. This increased sensitivity is susceptible to modification by known analgesic agents. The procedure used is based upon the method described by Randall and Selitto, Arch. Int. Pharmacodyn. l l l: 409 (1957). Rats were injected in the left hind paw with 0.1 ml. of a percent brewers yeast suspension. One hour later the drugs were administered orally via stomach tube. The pain threshold was measured one hour after drug administration by applying a steadily increasing pressure of 16 grams/second to the inflamed paw. The end point (pain threshold) was defined as the pressure necessary to cause the animal to struggle and/or vocalize.

Effective analgesic doses were obtained by determining the amount of drug required to cause a 50 percent increase in the mean pain threshold over that of parallel groups of control animals. The following values were obtained:

Table 6 Analgesic Activity of Anti-Inflammatory Agents (Pressure-Pain Method) Analgesic Dose Drug (mg/kg.)

A irin I85 NCll4O 72 Phenylbutazone 8.2 lndomethacin 1.]

494 (1956). Mice were administered the drug orally thirty minutes prior to the intraperitoneal injection of P.P.Q. Peak incidence of writhing occurs 5 minutes after injection of the irritant. At this time, the animals Table 7 Anti-writhing Activity of Anti-Inflammatory Agents ED,-,., vs. Writhing Drug (mg/kg.)

Aspirin 41 NC-l I40 20 Phcnylbutazone 49 lndomethacin 0.44

C. Rat Tail-Flick The method of DAmour and Smith, J. Pharmacol. Exptl. Therap. 72: 74 194] was adapted as an adjunct to the study of the analgesic properties of drugs. Briefly, the tails of rats are placed in an apparatus which automatically initiates a time mechanism and starts a heating coil which transmits a painful stimulus to the ventral surface of the tail. At the end point, the animal flicks its tail from the apparatus. Measurements are made either /2 and 1 hour post drug or one and 2 hours after drug administration, and averaged. Analgesia" may be considererd as a significant increase in the latency of pain perception and/or reaction time. The method is effective in detecting analgesic properties of the narcotic class of drugs (morphine, codeine, meperidine, etc.). Non-narcotic analgesics (aspirin, phenylbutazone, indomethacin, etc.) are not usually detected by this procedure. The test, therefore, may be used to differentiate between possible narcotic and non-narcotic analgesic activity. A doubling of the reaction time over control animals (no drug) may be considered significant. The following results were obtained with selected oral doses of representative narcotic analgesics and with high doses of certain nonnarcotic analgesics.

Table 8 Effect of Various Analgesics Thus, NC-l appears similar to aspirin and other related non-narcotic analgesics.

EXAMPLE 4 AntiSecretory Properties of NC-l 140 The purpose of these experiments was to ascertain whether NC-l l40 stimulated gastric secretion or caused significant changes in either free or total acid Table 9 AntiSccretory Activity of NC-l 140 LP. as Compared with Aspirin Table 10 Anti-Secretory Activity of NC-l 140 Orally Treatment Total Fluid /1 Reduction (Oral) Volume (m1.) from control Methylcellulose 3.4

(control) (100 mgjkg.)

NC-l 140 1.3 62

(200 mg./kg.)

Because NC-l 140 showed anti-secretory properties following both oral and intraperitoneal administration. additional studies were performed to compare this compound with several standard reference agents having known anti-inflammatory or anti-secretory activity.

Treatment T )tal Fluid Acid C1111 t m ml. HCl

(LP vojume (mm ln theses experiments, the stomachs of rats (fasted 48 hours) were ligated at the pylorus, followed immediy i 45 ately by the subcutaneous injection of various dose lev ifs iiig 4 4 61S 01 the selected drugs. After 4 hours the animals rlnfij/kg.) 4 I0 7 s were sacrificed and the total liquid volume, free and (Zoom/kg) total acid content, and pH of the gstnc ]U1CS were measured.

Table 11 Effect of Various Anti-Inflammatory and Anti-Secretory Drugs on Gastric Secretions in the Rat S.C. No. Liquid Dose of Volume Acid Content (mg/ml HCl) Drug (mg/kg.) rats (ml. 1 Free Total pH Methylcellulose (controls) 53 4.3 3.9 5.1 1.44 NC-1l40 12.5 8 2.7 2.8 4.1 1.52

25 8 2.6 2.9 4.3 1.50 8 1.3 1.6 3.6 1.56" Phenylbutazone 12.5 8 3.9 4.6 5.4 1.50 25 8 4.3 4.7" i 5.6 1.46 50 8 3.3 v 4.7 5.4 1.45 Aspirin 12.5 7 4.0 4.8 6.1" 1.46 25 7 3.4 4.4 5.9" 1.44 50 7 3.6 4.1 5.6 1.43 lndomethacin 1.25 8 4.8 4.5 5.5 1.47 2.5 8 4.1 3.1 4.9 1.43 5 8 3.3 3.7 5.1 1.47 Dimenhydrinate 12.5 8 4.5 4.3 5.4 1.45 (Dramamine) 25 8 3.5 4.0 4.9 1.42 50 8 2.6 3.6 4.5 1.50 Propantheline 0.05 8 3.4 3.3 4.9 1.37 (Pro-Banthine) 0.1 8 2.6 3.5 5.1 1.37

0.2 8 1.6 3.7 4.7 1.55 Trihexyphenidyl 12.5 8 2.9 3.8 4.8 1.49 (Artane) 25 8 2.5 3.5 4.4" 1.59 5O 8 1.5 3.3 3.8 1.63"

Based upon these data it appeared that NC-l 140 was not only devoid of gastric secretion stimulating properties but caused a marked reduc tion (47 percent) in total fluid volume. Aspirin was inactive in this respect.

1n addition. both NC-l 140 and aspirin caused reductions in both free and total acid content of the gastric juices.

The indication that NC-l 140 possessed antisecretory properties stimulated a similar study to determine if anti-secretory activity was also evident after oral administration. The indentical experimental procedure was repeated. with the exception that N01 140 was administered orally one hour prior to ligation.

Significant reduction (PQIOI from mean control value Significant increase (P201) from mean control value 13 EXAMPLE 5 Toxicological Evaluation of NC-1 140 A. Mortality Table 14 Continued Anti-inflammatory Activity Determined by Carrageenin-indueed Edema Method A Compound ED mgJkg. Comparative determination of acute (48 hour) and 5 5U delayed (5 day) lethality after single doses in rats and "l lggzggiizg mice with NO] 140 and other anti-inflammatory agents 3-ehlort i-s-t riazolo-l3,4-aleave the following data for the 50 percent lethal dose *l 60 L D 3.(I'LllCl'llUTO-S-ll'lllZUlO-l 3.4-all fitllisoquinolinc approx. fill Table 12 Comparative Determination of Acute and Delayed Lethality Oral sn) g/kg.) (L m (mg/kg.) Rats Mice Rats Mice Drug 48 hr. 5 Day 48 hr. 5 Day 48 hr. 5 Day 48 hr. 5 Day NC- l l40 I497 I368 I707 l53l 487 487 6l6 440 Aspirin 1857 1857 1900 1 900 l l29 80l 352 294 Phenylbutazone 618 599 1078 1078 264 2l2 283 277 liidomethacin 240 l7 160 45 100 39 80 32 Following single oral administration to both rats and 3-styryl s-til'iazorllozjl3.4;a]- d 160 i lSOqLllnD ll'lC y l'OC On 8 approx. mice, NC 1 140 appears reas onablyfree of toxicity. Via 34mmthio s triazolo I3,44]] oral or intraperitoneal administration in rats or mice, 25, isoquimiine 31 the agent appears comparably non-toxic or safer than gg z g 78 the other reference agents studied. Unlike indometha- 5 cin, NC-l 140 does not cause marked incidence of deisn i t I 3 4 layed day) deaths. B. Thera eutic Safet Index 3-meth'l-fi-bromo-s-triazolo-[3.441]- p y 0 Calculation of the therapeutic index, defined as the f l? I Approx 7-bi'omo-s-triazolo-l3.4-alvalue derived by dividing the 5 day rat oral LD value isoquinoline approx. by the oral ED value against carrageenin edema, re- -P 9- l th f H isoquinolirtc PP I00 vels e O Owmg- 3-phenyl-s-triazolo-l3.4-al- 99 isoquinoline Table 13 3-methyl-s-triazolo-[3,4-alisoquinoline I approx. S s-triazolo-l3,4-aI-isoquino inc approx. Oral Therapeutic lndices l D Therapeutic index isoquinolme approx. 43

3-trifluoromethyl-s-triazolo-[ 3,4-a N01 140 65 Phenylhumwnc 20 isoquinoline hydrochloride 34 Aspirin 8.3 liidomethacin EXAMPLE 7 Thus, NC-l 140 has a safety margin which is over 3 times that of phenylbutazone and 8 to 9 times that of aspirin and indomethacin.

EXAMPLE 6 Using the procedure set forth in Example 2A, the anti-inflammatory activity ofa number of s'tria2o1o-[3,4 al-isoquinoline compounds was determined. The results are set forth in the following table.

Table I4 Anti'inflammatory Activity Determined by Carrageenin-induced Edema Method Compound l-w g/ g 6-chloro-s-triazolo-[3,4-a]- approx. 20

isoquinoline 3 (N-morpholinylmethyl)-s-triazolo- [3,4-aI-isoquinoline dihydrochloride approx. 100 7-Hltl'O-S-ll'lillOlO-l3,4-211- isoquiiioline 62 o-methyl-s-triazolo-l3.4-a]-* isoquiiioline 44 6-cyanos-triazolo- [3.4a]-

isoquinolinc l07 o-carbamido-s-triazolo-[3,4-alisoquinoline approx. 30 obromo-s-triazolo-l3.4-:1 i 9 V. 11.

- fl Reduction of gastric secretion (nil/Wt) pm.)

Using the procedure set forth in Example 4, the antisecretory activity of a number of s-triazolo-[3,4-a] isoquinoline compounds was determined. The results are set forth in the following table.

utter subcutaneous administration to lasting nits int 50 nig./kg. unless otherwise indicated).

In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results attained.

As various changes could be made in the above methods without departing from the scope of the invention, it is intended that all matter contained in the above description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

What is claimed is:

l. A method of reducing inflammation in a mammal having such a disorder which comprises administering to said mammal an amount, sufficient to exert an antiinflammatory effect, of a compound selected from the group consisting of 3-trifluoromethyl-s-triazolo-[3,4- a]-isquinoline, B-(N-morpholinylmethyl)-s-triaz0lo- [3,4-a1-isoquin0line, 5-chl0r0-s-triaz0lo-[3,4-a]- isoquinoline, 7-nitro-s-triazol0-[3,4-a]-isoquinoline, 6-cyan0-s-triazol0-[3,4-a]-is0quinoline, -methoxy-striazol0-[3,4-a]-isoquinoline, 7-nitr0-3- trifluoromethyl-s-triazolo-l3,4-a]-isoquin0line, 3,6- dichlor0-s-triazol0-[3,4-a]-isoquin0line, 3-styryl striazolo-l3,4-aJ-is0quinoline, 3-methylthio-s-triazolo- [3,4-a]-isoquinoline, 3-ethylthio-s-triazolo-l 3 ,4-a]- isoquinoline, 8-methoxy-s-triazolo-[3,4-211- isoquinoline, 3-phenyl-s-triazol0-[3,4-a]-isoquinoline, striazolo-[3,4-al-isoquinoline, 3-oxo-s-triaz0lo-[3,4- a ]-isoquinoline, 3-thioxo-s-triaz0lo-[ 3,4-a1- isoquinoline, a 3-lower alkyl-s-triazolo-l3,4-a1- isoquinoline, a 3-lower alkyl-6-halo-s-triazo1o-[3,4-alisoquinoline, a 3-halo-s-triazolo-[3,4-a1-isoquinolinei a 6-halo-s-triazolo-[ 3,4-a ]-isoquin0line, a 7-halo-s triazolo-[ 3.4-a1-isoquinoline, 3-( N- morpholinylmethyl )-s-triazolo-[ 3 ,4-a] -isoq uinoline dihydrochloride, 3styryl-s-triazolo-[ 3,4-al-isoquinolinc hydrochloride and 3-trifluoromethyl-s-triazolo-I 3.4-a I- isoquinoline hydrochloride 2."lhe method ofclaim I wherein said compound is 3-trilluoromethyl-s-triazolo-| 3 4-a l-isoquinoline.

3. The method ofclaini I wherein said compound is o-chloro-s-triazolo-l -14-11I-isoquinolinc.

4. The method of claim I wherein said compound is 7-nitro-s-triazolo-l 3,4-a I-isoquinoline.

5. The method of claim 1 wherein said compound is 6-bromo-s-triazolo-I 3,4-a l-isoquinoline.

6. The method of claim 1 wherein said compound is 6methoxy-s-triaZolo-[3,4-a]-is0quinoline.

7. The method of claim 1 wherein said compound is 3-chl0ros-triaz0l0-[3,4-a]-isoquinoline.

-8. The method of claim 1 wherein said compound is 3,6-dichl0ro-s-triaz0lo-[3.4-a]-isoquin0line.

9. The method of claim 1 wherein said compound is 3-methylthio-s-triazolo-[ 3,4-a]-isoquinoline.

10. The method of claim 1 wherein said compound is 8-methoxy-s-triazolo-l 3,4-al-isoquinoline.

11. The method of claim 1 wherein said compound is 7-bromo-striazol0-I 3,4-al-isoquinoline.

12. The method of claim 1 wherein said compound is s-triaZolo-l 3,4-a]-isoquinoline.

13. The method of claim 1 wherein said compound is 3-oxo-s-triazolo-l3,4-a]-isoquinoline. 

1. A METHOD OF REDUCING INFLAMMATION IN A MAMMAL HAVING SUCH A DISORDER WHICH COMPRISES ADMINISTERING TO SAID MAMMAL AN AMOUNT, SUFFICIENT TO EXERT AN ANTI-INFLAMMATORY EFFECT, OF A COMPOUND SELECTED FROM THE GROUP CONSISTING OF 3TRIFLUOROMETHYL-S-TRIAZOLO-(3,4-A)-ISOQUINOLINE, 3-(NMORPHOLINYLETHYL)-S-TRIZOLO-(3,4-A)-ISOQUINOLINE, 5-CHLOROS-TRIAZOLO-(3,4-A)-ISOQUINOLINE, 7-NITRO-S-TRIAZOLO-(3,4-A)ISOQUINOLINE, 6-CYANO-S-TRIAZOLO-(3,4-A)-ISOQUINLOINE, 6METHOXY-S-TRIAZOLO-(3,4-A)-ISQUINOLINE, 7-NITRO-3TRIFLUOROMETHYL-S-TRIAZOLO-(3,4-A)-ISOQUINOLINE, 3,6-DICHLOROS-TRIAZOLO-(3,4-A)-ISOQUINOLINE, 3-STYRYL-S-TRIAZOLO-(3,4-A)ISOQUINOLINE, 3-METHYLTHIO-S-TRIAZOLO -(3,4-A)-IQUINOLINE, 3ETHYLTHIO-S-TRIAZOLO-(3,4-A)-ISOQUINOLINE, 8-METHOXY-STRIAZOLO-(3,4-A)-ISQUINOLINE, 3-PHENYL-S-TRIAZOLO-(3,4-A)ISQUINOLINE, S-TRIAZOLO-(3,4-A)-ISOQUINOLINE, 3-OXO-S-TRIAZOLO(3,4-A)-ISOQUINOLINE,3-TRIOXO-S-TRIAZOLO-(3,4-A) -ISOQUINOLINE, A 3-LOWER ALKYL-S-TRIAZOLO-(3,4-A)-ISOQUINOLINE, A 3-LOWER ALLKYL-6-HALO-S-TRIAZOLO-(3,4-A)-IWOQUINOLINE, A 3-HALO-S-TRIAZOLO(3,4-A)-ISOQUINOLINE, A 6-HALO-S-TRIAZLOL-(3,4-A)ISOQUINOLINE, A 7-HALO-S-TRIAZOLO-(3,4-A)-ISOQUINOLINE, 3-(NMORPHOLINYMETHYL)-S-TRIAZOLO-(3,4-A)-ISOQUINOLINE DIHYDROCHLORIDE, 3STYRYL-S-TRIAZOLO-(3,4-A)-ISOQUINOLINE HYDROCHLORIDE AND 3-TRIFLUOROMETHYL-S-TRIAZOLO-(3,4-A)-ISOQUINOLINE, CHLORIDE.
 2. The method of claim 1 wherein said compound is 3-trifluoromethyl-s-triazolo-(3,4-a)-isoquinoline.
 3. The method of claim 1 wherein said compound is 6-chloro-s-triazolo-(3,4-a)-isoquinoline.
 4. The method of claim 1 wherein said compound is 7-nitro-s-triazolo-(3,4-a)-isoquinoline.
 5. The method of claim 1 wherein said compound is 6-bromo-s-triazolo-(3,4-a)-isoquinoline.
 6. The method of claim 1 wherein said compound is 6-methoxy-s-triazolo-(3,4-a)-isoquinoline.
 7. The method of claim 1 wherein said compound is 3-chloro-s-triazolo-(3,4-a)-isoquinoline.
 8. The method of claim 1 wherein said compound is 3,6-dichloro-s-triazolo-(3,4-a)-isoquinoline.
 9. The method of claim 1 wherein said compound is 3-methylthio-s-triazolo-(3,4-a)-isoquinoline.
 10. The method of claim 1 wherein said compound is 8-methoxy-s-triazolo-(3,4-a)-isoquinoline.
 11. The method of claim 1 wherein said compound is 7-bromo-s-triazolo-(3,4-a)-isoquinoline.
 12. The method of claim 1 wherein said compound is s-triazolo-(3,4-a)-isoquinoline.
 13. The method of claim 1 wherein said compound is 3-oxo-s-triazolo-(3,4-a)-isoquinoline. 